Methods for improving muscle and heart function

ABSTRACT

The present invention encompasses combinations comprising a plurality of amino acids, formulations thereof for use in humans and animals, as well as methods for increasing muscle protein synthesis, muscle strength, and/or muscle function in a subject with chronic heart failure, cardiac cachexia or a combination thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present non-provisional utility application claims priority to U.S.Provisional Patent Application No. 61/814,034, entitled “Methods ForImproving Heart Function, filed on filed Apr. 19, 2013, and which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention encompasses a combination comprising a pluralityof amino acids, formulations thereof for use in humans and animals, aswell as methods for increasing muscle protein synthesis, musclestrength, and/or muscle function.

BACKGROUND OF THE INVENTION

The decline in muscle mass, strength and function that occurs withaging, especially in older persons with chronic heart failure, as wellas the impact of those changes on health and quality of life, is welldocumented. Whereas the effectiveness of resistance exercise training inimproving strength, is also clear, <30% of all adult North Americansexercise regularly, and 50% of those who start an exercise program dropout within the first six months. Additional approaches are necessary toeffectively ameliorate the loss of muscle mass and strength that occurswith aging.

Amino acids are potent stimulators of muscle protein synthesis in boththe young and elderly. However, the anabolic response to a mixed mealcontaining both amino acids and carbohydrates is diminished in elderlyindividuals. It is therefore possible that the loss of muscle mass andstrength that occurs with aging may be due, in part, to an intake ofprotein that is less than optimal. If this is the case, ingestion of anutritional supplement containing amino acids may be a practicalapproach to improving muscle mass and strength in the elderly.

SUMMARY OF THE INVENTION

One aspect of the invention encompasses a combination comprisingphenylalanine, valine, threonine, tryptophan, methionine, leucine,isoleucine, lysine, histidine, citrulline and L-carnitine in a w/w ratioof about 4:about 6:about 5:about 2:about 2:about 25:about 7:about10:about 1:about 8:about 3, respectively.

Another aspect of the invention comprises a combination listed in TableA, Table B, or Table C.

Another aspect of the invention comprises a method for increasing muscleprotein synthesis, increasing muscle strength, increasing musclefunction, or a combination thereof, the method comprising administeringto a subject a combination comprising phenylalanine, valine, threonine,tryptophan, methionine, leucine, isoleucine, lysine, histidine,citrulline and L-carnitine in a w/w ratio of about 4:about 6:about5:about 2:about 2:about 25:about 7:about 10:about 1:about 8:about 3,respectively. A subject may have chronic heart failure, cardiac cachexiaor a combination thereof. Alternatively a subject may be at risk formuscle atrophy.

Another aspect of the invention comprises a method for increasing muscleprotein synthesis, increasing muscle strength, increasing musclefunction, or a combination thereof, the method comprising administeringto a subject a combination listed in Table A, Table B or Table C. Asubject may have chronic heart failure, cardiac cachexia or acombination thereof. Alternatively a subject may be at risk for muscleatrophy.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts graphs showing a combination of essential amino acidsincreases net protein synthesis in the leg muscle of healthy humanvolunteers over the age of 65 years. The y-axis depicts the increaseabove basal in the balance between protein synthesis and breakdown (netprotein synthesis). Volunteers ingested 6.7 grams of a mixture of EAAsin the profile of whey protein (Normal Leucine) or a mixture of the sameamount of EAAs with 40% leucine (High Leucine), with the proportionatecontribution of the other EAAs decreased. Muscle protein synthesis wasmeasured using a stable isotope tracer technique that was based on bloodsampling from indwelling catheters in the femoral artery and vein andmuscle tissue sampling by needle biopsy.

FIG. 2 depicts a graph showing the percent (%) increase in muscleprotein synthesis in response to 15 grams of whey protein isolate, anEAA combination (Table C), or 115 g (4 oz.) of beef containing 39 gramsprotein. Protein synthesis was measured as described above.

FIG. 3 depicts graphs showing that dietary supplementation with acombination of EAAs (Table C) ameliorated the detrimental effects ofstrict bed rest on parameters of physical function. Physical functionwas measured using basic standardized tests, including floor transfer(A), stair ascent (B), and standing plantar flexior (C).

FIG. 4 depicts a graph showing the increase in net protein synthesisabove base (y-axis) in the leg muscle of normal volunteers after astrenuous resistance exercise (left), after intake of amino acids(middle), or both (right). In these experiments, subjects wereadministered a commercially-available intravenous formulation of aminoacids (Travasol).

FIG. 5 depicts plasma arginine responses (A) and nitric oxide (NO)synthesis rates (B) in basal period and during ingestion of 3 g ofcitrulline (CIT) sip ingestion over 3 hours in elderly (n=8, 77±9y, 93±4kg) and young individuals (n=8, 27±9y, 70±5 kg). NO synthesis rates areexpressed per kg fat free mass. *Significantly different from theelderly, p<0.05; † Significantly different from basal period, p<0.05.

FIG. 6 depicts graphs showing leg muscle protein synthesis (umolphenylalanine/100 g tissue/h) in rabbits during an infusion of abalanced mixture of amino acids (Travasol, 1.5 ml/kg/h) or the sameamino acid solution with extra arginine added Arg-Travasol (Travasol at0.75 ml/kg/h; arginine at 205 umol/kg/h). *Significantly different fromArg-Travasol, p<0.05.

FIG. 7 depicts graphs showing liver fat in elderly human volunteers(n=16) at baseline and after 8 and 16 weeks of therapy with two doses ofa combination of EAAs (11 g per dose; Table C) each day. Liver fat wasmeasured using magnetic resonance spectroscopy (MRS). Units of liver fatare expressed in relation to a standard of Intralipid. The reduction inliver fat was associated with an improvement in insulin sensitivity.

FIG. 8 depicts graphs showing carnitinepalmitoyltransferase (CPT)-1activity decreases with age in mitochondria (2 vs 18 mos, p<0.004).Treatment with oral propionyl-L-carnitine (PLC) in old hearts restoredCPT to that of young hearts (p<0.02) n=6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a combination comprising a plurality ofamino acids. When administered to a subject, a combination of theinvention produces an effective response in the subject. An effectiveresponse can be an increase in muscle protein synthesis, an increase inmuscle strength, an increase in muscle function, or a combinationthereof. Also contemplated are compositions comprising a combination ofthe invention formulated for use in humans and animals. Advantageously,a combination of the invention produces an effective response whenadministered to a subject, even in the absence of an exercise program.Additional aspects to the invention are described in further detailbelow.

I. Combination Comprising a Plurality Amino Acids

The present invention provides a combination comprising a plurality ofamino acids. An amino acid can be a proteinogenic amino acid or anon-proteinogenic amino acid. In an aspect, a plurality of amino acidscomprises two or more proteinogenic amino acids and, optionally, one ormore non-proteinogenic amino acids. The proteinogenic amino acids arephenylalanine, valine, threonine, tryptophan, methionine, leucine,isoleucine, lysine, histidine, alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, ornithine, proline,selenocysteine, serine and tyrosine. A combination of the invention maycomprise any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or 22 proteinogenic amino acids. Anon-proteinogenic amino acid does not participate in peptide synthesis,and may or may not be naturally occurring. The non-proteinogenic aminoacids far outnumber the proteinogenic amino acids, approaching at least300. See, for example, Peptides from A to Z: A Concise Encyclopedia,2008, Wiley-VCH Verlag GmbH & Co. Combinations comprising anon-proteinogenic amino acid may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 or more non-proteinogenic amino acids.

In another aspect, a plurality of amino acids comprises one or moreessential amino acids. Applicants have found the preferential use ofessential amino acids compared to non-essential amino acids in acombination of the invention reduces the amount of amino acids that mustbe given to elicit an effective response. An essential amino acid (EAA)is an amino acid that cannot be synthesized de novo by a subject, andtherefore must be supplied in its diet. The amino acids regarded asessential for humans are phenylalanine, valine, threonine, tryptophan,methionine, leucine, isoleucine, lysine, and histidine. In someembodiments, a combination of the invention comprises phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,and histidine.

When a combination of the invention comprises all nine essential aminoacids, the nine essential amino acids are at least about 80% of thetotal weight of the combination. For example, the nine essential aminoacids can be at least about 80%, at least about 81%, at least about 82%,at least about 83%, at least about 84%, at least about 85%, at leastabout 86%, at least about 87%, at least about 88%, at least about 89%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94, at least about 95, at least about 96%, atleast about 97%, at least about 98%, at least about 99% of the totalweight of the combination. Alternatively, the nine essential amino acidscan be about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94, about 95, about 96%, about 97%, about 98%,about 99%, or about 100% of the total weight of the combination. Inanother alternative, the nine essential amino acids can be about 80% toabout 90%, or about 90% to about 100% of the total weight of thecombination. In still another alternative, the nine essential aminoacids can be about 80% to about 95%, or about 85% to about 100% of thetotal weight of the combination. In a different alternative, the nineessential amino acids can be about 85% to about 95% of the total weightof the combination. In a still different alternative, the nine essentialamino acids can be about 80% to about 85%, about 85% to about 90%, about90% to about 95%, or about 95% to about 100% of the total weight of thecombination. In yet another alternative, the nine essential amino acidscan be about 80% to about 100% of the total weight of the combination.

In another aspect, a combination of the invention has a high proportionof leucine compared to the total amount of all the EAAs in thecombination. In some embodiments, the percentage of leucine is about 30%to about 40% by weight of all the EAAs. In a preferred embodiment, thepercentage of the total EAAs that is leucine is about 35%, about 36%,about 37%, about 38%, about 39% or about 40% by weight of all the EAAs.In another preferred embodiment, the percentage of the total EAAs thatis leucine is about 35% to about 40% by weight of all the EAAs.

In another aspect, a plurality of amino acids comprises one or moreessential amino acids, and one or more conditionally essential aminoacids and/or one or more conditionally essential amino acid precursors.The amino acids arginine, cysteine, glycine, glutamine, proline, serineand tyrosine are considered conditionally essential, meaning they arenot normally required in the diet, but must be supplied exogenously tospecific populations that do not synthesize them in adequate amounts.For example, the body produces sufficient arginine to satisfy metabolicrequirements under normal conditions. Therefore, argininesupplementation is not necessary to stimulate muscle protein synthesisfor many subjects. However, in certain clinical circumstances, includingin elderly with heart failure, endogenous arginine production isinadequate to meet all demands. For such a population, it isadvantageous for combinations of the invention to comprise arginine, ora precursor of the amino acid arginine. The term “amino acid precursor”refers to a metabolic precursor of an amino acid. For example, serine isa metabolic precursor of cysteine and glycine; and 3-phosphoglycerate isa metabolic precursor of serine and, therefore, of cysteine and glycinetoo. As a second example, citrulline is a metabolic precursor ofarginine. The metabolic pathways that synthesize amino acids are wellknown in the art, and a skilled artisan may refer to these to identifymetabolic precursors of other amino acids. In some embodiments, acombination of the invention comprises phenylalanine, valine, threonine,tryptophan, methionine, leucine, isoleucine, lysine, histidine, and atleast one conditionally essential amino acid. For example, thecombination may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conditionallyessential amino acids. In other embodiments, a combination of theinvention comprises phenylalanine, valine, threonine, tryptophan,methionine, leucine, isoleucine, lysine, histidine, and at least oneconditionally essential amino acid precursor. For example, thecombination may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conditionallyessential amino acid precursors. In still other embodiments, acombination of the invention comprises phenylalanine, valine, threonine,tryptophan, methionine, leucine, isoleucine, lysine, histidine, at leastone non-essential amino acid and at least one conditionally essentialamino acid precursor. In a preferred embodiment, a combination of theinvention comprises citrulline.

When a combination of the invention comprises one or more conditionallyessential amino acid and/or conditionally essential amino acidprecursor, the one or more amino acid and/or amino acid precursor is nomore than about 20% by weight of the combination. For example, acombination may comprise one or more conditionally essential amino acidsand/or amino acid precursors in an amount that is about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, or about 20% by weight of thecombination. In another alternative, the one or more conditionallyessential amino acids and/or conditionally essential amino acidprecursors can be about 1% to about 10%, or about 10% to about 20% ofthe total weight of the combination. In still another alternative, theone or more conditionally essential amino acids and/or conditionallyessential amino acid precursors can be about 1% to about 15%, or about5% to about 20% of the total weight of the combination. In a differentalternative, the one or more conditionally essential amino acids and/orconditionally essential amino acid precursors can be about 5% to about15% of the total weight of the combination. In a still differentalternative, the one or more conditionally essential amino acids and/orconditionally essential amino acid precursors can be about 1% to about5%, about 5% to about 10%, about 10% to about 15%, or about 15% to about20% of the total weight of the combination.

In another aspect, a plurality of amino acids comprises one or morenon-standard amino acid. As used herein, the term “non-standard aminoacid” refers to a non-proteinogenic amino acid that occurs naturally ina cell. In a cell, a non-standard amino acid is generated bymodification of a proteinogenic amino acid. Non-limiting examples ofnon-standard amino acids include L-carnitine, GABA, hydroxyproline,hydroxylysine, pyrrolysine, selenomethionine, hypusine, L-DOPA,2-aminobutyric acid, dehydralanine, gamma-carboxyglutamic acid. In someembodiments, a combination of the invention comprises phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, and at least non-standard amino acid. For example, thecombination may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-standardamino acids. In other embodiments, a combination of the inventioncomprises (i) phenylalanine, valine, threonine, tryptophan, methionine,leucine, isoleucine, lysine, and histidine; (ii) at least oneconditionally essential amino acid; and (iii) at least one non-standardamino acid. In still other embodiments, a combination of the inventioncomprises (i) phenylalanine, valine, threonine, tryptophan, methionine,leucine, isoleucine, lysine, and histidine; (ii) at least oneconditionally essential amino acid precursor; and (iii) at least onenon-standard amino acid. In yet other embodiments, a combination of theinvention comprises (i) phenylalanine, valine, threonine, tryptophan,methionine, leucine, isoleucine, lysine, and histidine; (ii) at leastone conditionally essential amino acid; (iii) at least one conditionallyessential amino acid precursor; and (iv) at least one non-standard aminoacid. In preferred embodiments, the conditionally essential amino acidis citrulline and the non-standard amino acid is L-carnitine.

When a combination of the invention comprises one or more non-standardamino acid, the one or more non-standard amino acid is no more thanabout 20% by weight of the combination. For example, a combination maycomprise one or more non-standard amino acid in an amount that is about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% byweight of the combination. In another alternative, the one or morenon-standard amino acid can be about 1% to about 10%, or about 10% toabout 20% of the total weight of the combination. In still anotheralternative, the one or more non-standard amino acid can be about 1% toabout 15%, or about 5% to about 20% of the total weight of thecombination. In a different alternative, the one or more non-standardamino acid can be about 5% to about 15% of the total weight of thecombination. In a still different alternative, the one or morenon-standard amino acid can be about 1% to about 5%, about 5% to about10%, about 10% to about 15%, or about 15% to about 20% of the totalweight of the combination.

In a preferred embodiment, a combination of the invention comprisesphenylalanine, valine, threonine, tryptophan, methionine, leucine,isoleucine, lysine, and histidine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1,respectively. In another preferred embodiment, a combination of theinvention comprises phenylalanine, valine, threonine, tryptophan,methionine, leucine, isoleucine, lysine, and histidine in a w/w ratio ofabout 4:about 6:about 5:about 2:about 2:about 25:about 7:about 10:about1, respectively, wherein the nine essential amino acids are at leastabout 80% of the total weight of the combination. In another preferredembodiment, a combination of the invention comprises phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine and citrulline in a w/w ratio of about 4:about 6:about 5:about2:about 2:about 25:about 7:about 10:about 1:about 8, respectively. Inyet another preferred embodiment, a combination of the inventioncomprises phenylalanine, valine, threonine, tryptophan, methionine,leucine, isoleucine, lysine, histidine and citrulline in a w/w ratio ofabout 4:about 6:about 5:about 2:about 2:about 25:about 7:about 10:about1:about 8, respectively, wherein the ten amino acids are at least about95% of the total weight of the combination. In still another preferredembodiment, a combination of the invention comprises phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively. In an exemplary embodiment, a combination ofthe invention is listed in Table A.

TABLE A AMINO ACID AMOUNT Histidine 0.20 g Isoleucine 1.35 g Leucine5.08 g Lysine 2.12 g Methionine 0.42 g Phenylalanine 0.85 g Threonine1.17 g Valine 1.39 g Tryptophan 0.42 g Citrulline 1.50 g L-Carnitine0.50 g

Amino acids may be supplied as peptides of intact protein or,preferably, in their free form. If not obtained commercially, individualamino acids may be produced by methods well known in the art, includingchemical synthesis or use of recombinant microorganisms. A skilledartisan will appreciate that all amino acids except glycine may exist inmore than one isomeric form—specifically, as a D-isomer or an L-isomer.Combinations of the present invention are preferably comprised of Lamino acids.

In another aspect, combinations of the invention may further compriseone or more additional nutrients. The term “nutrient”, as used herein,refers to prebiotics, vitamins, carbohydrates, fiber, fatty acids,sulfates, minerals, antioxidants, and other food ingredients used insubject's metabolism which are taken in from its environment. Suitablevitamins may include, but are not limited to: vitamin B1, vitamin B2,vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, lipoicacid, vitamin A, biotin, vitamin K, vitamin C, vitamin D, and vitamin E.Suitable minerals may include, but are not limited to compoundscontaining: iron, copper, magnesium, manganese, molybdenum, nickel, andzinc. Suitable enzyme cofactors may include, but are not limited to:adenosine triphosphate (ATP), S-adenosyl methionine (SAM), coenzyme B,coenzyme M, coenzyme Q, glutathione, heme, methanofuran, and nucleotidesugars. Suitable lipids may include, but are not limited to, fattyacids, glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenol lipids, saccharolipids and polyketides. Non-limitingexamples of fatty acids include myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, arachidonic acid, eicosapentaenoic acid, erucic acid,docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, behenic acid,lignoceric acid, cerotic acid, hexadecatrienoic acid, alpha-linolenicacid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid,heneicosapentaenoic acid, docosapentaenoic acid, clupanodonic acid,docasehaenoic acid, tetracosapentaenoic acid, and tetracosahexaenoicacid. Additional non-limiting examples of nutrients may include Thiamin,Riboflavin, Niacin, Folate, Pantothenic acid, Calcium, Phosphorus,Magnesium, Manganese, Iron, Zinc, Copper, Selenium, Sodium, Potassium,betacarotene, retinol, alphatocopherol, betatocopherol, gammatocopherol,deltatocopherol, alphatoctrienol, betatoctrienol, gammatocotrienol,deltatocotrienol, apo-8-carotenal, trans-lycopene, cis-lycopene,trans-beta-carotene, and cis-beta-carotene, caffeine. In someembodiments, combinations of the invention further comprise onenutrient. In other embodiments, combinations of the invention furthercomprise at least one nutrient. For example, a combination of theinvention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nutrients.In still other embodiments, combinations of the invention furthercomprise two or more nutrients. In preferred embodiments, combinationsof the invention further comprise at least one nutrient selected fromthe group consisting of omega-3 fatty acid and biotin. In otherpreferred embodiments, combinations of the invention further comprisetwo nutrients selected from the group consisting of omega-3 fatty acidand biotin. The overall contribution of the one or more nutrients to thetotal weight of the combination is substantially less than thecontribution of the plurality of amino acids. Generally, the one or morenutrients comprise no more than about 10% by weight, preferably no morethan about 5% by weight, more preferably no more than about 3% by weightof the combination. In preferred embodiments, the one or more nutrientis biotin. In other preferred embodiments, the one or more nutrient isone or more omega-3 fatty acid. In other preferred embodiments, the oneor more nutrient is biotin and one or more omega-3 fatty acid. In anexemplary embodiment, a combination of the invention comprising one ormore nutrient is listed in Table B or Table C.

TABLE B COMPONENT AMOUNT Histidine 0.20 g Isoleucine 1.35 g Leucine 5.08g Lysine 2.12 g Methionine 0.42 g Phenylalanine 0.85 g Threonine 1.17 gValine 1.39 g Tryptophan 0.42 g Citrulline 1.50 g L-carnitine 0.50 gBiotin 150 μg Omega-3 fatty acids 450 mg

TABLE C COMPONENT AMOUNT Histidine 0.20 g Isoleucine 1.35 g Leucine 5.08g Lysine 2.12 g Methionine 0.42 g Phenylalanine 0.85 g Threonine 1.17 gValine 1.39 g Tryptophan 0.42 g Biotin 150 μg Omega-3 fatty acids 450 mgII. Formulations

In each of the above embodiments, amino acids and nutrients (whenpresent) may be formulated for animal or human use. In some embodiments,each amino acid and nutrient (when present) is formulated separately. Inother embodiments, two or more amino acids and nutrients (when present)are formulated together. In still other embodiments, all the amino acidsand nutrients comprising a combination of the invention are formulatedtogether. The one or more formulations may then be processed into one ormore dosage forms that can be administered together, sequentially, orover a period of time (for example, over 1 minute, 10 minutes, 30minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24hours, or more). Administration can be performed using standardeffective techniques, including oral, parenteral (e.g. intravenous,intraperitoneal, subcutaneous, intramuscular), buccal, sublingual, orsuppository administration. The term orally, as used herein, refers toany form of administration by mouth, including addition of a compositionto animal feed or other food product. Formulation of pharmaceuticalcompositions is discussed in, for example, Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), andLiberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y. (1980).

Methods of preparing compositions for animal or human use are well knownin the art. For instance, a composition may be generally formulated as aliquid composition, a solid composition or a semi-solid composition.Liquid compositions include, but are not limited to, aqueoussuspensions, solutions, emulsions, elixirs, or syrups. Liquidcomposition will typically include a solvent carrier selected from apolar solvent, a non-polar solvent, or a combination of both. The choiceof solvent will be influenced by the properties of the components of thecomposition. For example, if the components are water-soluble, a polarsolvent may be used. Alternatively, if the components of the compositionare lipid-soluble, a non-polar solvent may be used. Suitable polar andnon-polar solvents are known in the art. Semi-solid compositions includedouches, suppositories, creams, and topicals. Dry compositions include,but are not limited to, reconstitutable powders, chewable tablets, quickdissolve tablets, effervescent tablets, multi-layer tablets, bi-layertablets, capsules, soft gelatin capsules, hard gelatin capsules,caplets, lozenges, chewable lozenges, beads, powders, granules,particles, microparticles, and dispersible granules. Formulations mayinclude a combination of the invention along with an excipient.Non-limiting examples of excipients include binders, diluents (fillers),disintegrants, effervescent disintegration agents, preservatives(antioxidants), flavor-modifying agents, lubricants and glidants,dispersants, coloring agents, pH modifiers, chelating agents,antimicrobial agents, release-controlling polymers, and combinations ofany of these agents.

Non-limiting examples of binders suitable for the formulations ofvarious embodiments include starches, pregelatinized starches, gelatin,polyvinylpyrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohols,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof. The polypeptidemay be any arrangement of amino acids ranging from about 100 to about300,000 Daltons. In one embodiment, the binder may be introduced intothe mixture to be granulated in a solid form including but not limitedto a crystal, a particle, a powder, or any other finely divided solidform known in the art. In another embodiment, the binder may bedissolved or suspended in a solvent and sprayed onto the mixture in agranulation device as a binder fluid during granulation.

Non-limiting examples of diluents (also referred to as “fillers” or“thinners”) include carbohydrates, inorganic compounds, andbiocompatible polymers, such as polyvinylpirrolydone (PVP). Othernon-limiting examples of diluents include dibasic calcium sulfate,tribasic calcium sulfate, starch, calcium carbonate, magnesiumcarbonate, microcrystalline cellulose, dibasic calcium phosphate,tribasic calcium phosphate, magnesium carbonate, magnesium oxide,calcium silicate, talc, modified starches, saccharides such as sucrose,dextrose, lactose, microcrystalline cellulose, fructose, xylitol, andsorbitol, polyhydric alcohols; starches; pre-manufactured directcompression diluents; and mixtures of any of the foregoing.

Disintegrents may be effervescent or non-effervescent. Non-limitingexamples of non-effervescent disintegrants include starches such as cornstarch, potato starch, pregelatinized and modified starches thereof,sweeteners, clays, such as bentonite, micro-crystalline cellulose,alginates, sodium starch glycolate, gums such as agar, guar, locustbean, karaya, pecitin, and tragacanth. Suitable effervescentdisintegrants include but are not limited to sodium bicarbonate incombination with citric acid, and sodium bicarbonate in combination withtartaric acid.

Non-limiting examples of preservatives include, but are not limited to,ascorbic acid and its salts, ascorbyl palmitate, ascorbyl stearate,anoxomer, N-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid,o-aminobenzoic acid, p-aminobenzoic acid (PABA), butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid,canthaxantin, alpha-carotene, beta-carotene, beta-caraotene,beta-apo-carotenoic acid, carnosol, carvacrol, catechins, cetyl gallate,chlorogenic acid, citric acid and its salts, clove extract, coffee beanextract, p-coumaric acid, 3,4-dihydroxybenzoic acid,N,N′-diphenyl-p-phenylenediamine (DPPD), dilauryl thiodipropionate,distearyl thiodipropionate, 2,6-di-tert-butylphenol, dodecyl gallate,edetic acid, ellagic acid, erythorbic acid, sodium erythorbate,esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethylgallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA),eucalyptus extract, eugenol, ferulic acid, flavonoids (e.g., catechin,epicatechin, epicatechin gallate, epigallocatechin (EGC),epigallocatechin gallate (EGCG), polyphenol epigallocatechin-3-gallate),flavones (e.g., apigenin, chrysin, luteolin), flavonols (e.g.,datiscetin, myricetin, daemfero), flavanones, fraxetin, fumaric acid,gallic acid, gentian extract, gluconic acid, glycine, gum guaiacum,hesperetin, alpha-hydroxybenzyl phosphinic acid, hydroxycinammic acid,hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid,hydroxytryrosol, hydroxyurea, rice bran extract, lactic acid and itssalts, lecithin, lecithin citrate; R-alpha-lipoic acid, lutein,lycopene, malic acid, maltol, 5-methoxy tryptamine, methyl gallate,monoglyceride citrate; monoisopropyl citrate; morin,beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate,oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine,phosphoric acid, phosphates, phytic acid, phytylubichromel, pimentoextract, propyl gallate, polyphosphates, quercetin, trans-resveratrol,rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin,sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaricacid, thymol, tocopherols (i.e., alpha-, beta-, gamma- anddelta-tocopherol), tocotrienols (i.e., alpha-, beta-, gamma- anddelta-tocotrienols), tyrosol, vanilic acid,2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., Ionox 100),2,4-(tris-3′,5′-bi-tert-butyl-4′-hydroxybenzyl)-mesitylene (i.e., Ionox330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxy butyrophenone,tryptamine, tyramine, uric acid, vitamin K and derivates, vitamin Q10,wheat germ oil, zeaxanthin, or combinations thereof. In an exemplaryembodiment, the preservatives is an antioxidant, such as a-tocopherol orascorbate, and antimicrobials, such as parabens, chlorobutanol orphenol.

Suitable flavor-modifying agents include flavorants, taste-maskingagents, sweeteners, and the like. Flavorants include, but are notlimited to, synthetic flavor oils and flavoring aromatics and/or naturaloils, extracts from plants, leaves, flowers, fruits, and combinationsthereof. Other non-limiting examples of flavors include cinnamon oils,oil of wintergreen, peppermint oils, clover oil, hay oil, anise oil,eucalyptus, vanilla, citrus oils such as lemon oil, orange oil, grapeand grapefruit oil, fruit essences including apple, peach, pear,strawberry, raspberry, cherry, plum, pineapple, and apricot.

Taste-masking agents include but are not limited to cellulosehydroxypropyl ethers (HPC) such as Klucel®, Nisswo HPC and PrimaFloHP22; low-substituted hydroxypropyl ethers (L-HPC); cellulosehydroxypropyl methyl ethers (HPMC) such as Seppifilm-LC, Pharmacoat®,Metolose SR, Opadry YS, PrimaFlo, MP3295A, Benecel MP824, and BenecelMP843; methylcellulose polymers such as Methocel® and Metolose®;Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel®,Aqualon®-EC, Surelease; Polyvinyl alcohol (PVA) such as Opadry AMB;hydroxyethylcelluloses such as Natrosol®; carboxymethylcelluloses andsalts of carboxymethylcelluloses (CMC) such as Aualon®-CMC; polyvinylalcohol and polyethylene glycol co-polymers such as Kollicoat IRO;monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols,modified food starch, acrylic polymers and mixtures of acrylic polymerswith cellulose ethers such as Eudragit® EPO, Eudragit® RD100, andEudragit® E100; cellulose acetate phthalate; sepifilms such as mixturesof HPMC and stearic acid, cyclodextrins, and mixtures of thesematerials. In other embodiments, additional taste-masking agentscontemplated are those described in U.S. Pat. Nos. 4,851,226, 5,075,114,and 5,876,759, each of which is hereby incorporated by reference in itsentirety.

Non-limiting examples of sweeteners include glucose (corn syrup),dextrose, invert sugar, fructose, and mixtures thereof (when not used asa carrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; sugar alcohols such as sorbitol, mannitol,sylitol, hydrogenated starch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

Lubricants may be utilized to lubricate ingredients that form acomposition of the invention. As a glidant, the lubricant facilitatesremoval of solid dosage forms during the manufacturing process.Non-limiting examples of lubricants and glidants include magnesiumstearate, calcium stearate, zinc stearate, hydrogenated vegetable oils,sterotex, polyoxyethylene monostearate, talc, polyethylene glycol,sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, andlight mineral oil. The composition will generally comprise from about0.01% to about 10% by weight of a lubricant. In some embodiments, thecomposition will comprise from about 0.1% to about 5% by weight of alubricant. In a further embodiment, the composition will comprise fromabout 0.5% to about 2% by weight of a lubricant.

Dispersants may include but are not limited to starch, alginic acid,polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified woodcellulose, sodium starch glycolate, isoamorphous silicate, andmicrocrystalline cellulose as high hydrophilic-lipophilic balance (HLB)emulsifier surfactants.

Depending upon the embodiment, it may be desirable to include a coloringagent. Suitable color additives include but are not limited to food,drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), orexternal drug and cosmetic colors (Ext. D&C). These colors or dyes,along with their corresponding lakes, and certain natural and derivedcolorants may be suitable for use in various embodiments.

Non-limiting examples of pH modifiers include citric acid, acetic acid,tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid,sorbic acid, benzoic acid, sodium carbonate and sodium bicarbonate.

A chelating agent may be included as an excipient to immobilizeoxidative groups, including but not limited to metal ions, in order toinhibit the oxidative degradation of the morphinan by these oxidativegroups. Non-limiting examples of chelating agents include lysine,methionine, glycine, gluconate, polysaccharides, glutamate, aspartate,and disodium ethylenediaminetetraacetate (Na²EDTA).

An antimicrobial agent may be included as an excipient to minimize thedegradation of the compound according to this disclosure by microbialagents, including but not limited to bacteria and fungi. Non-limitingexamples of antimicrobials include parabens, chlorobutanol, phenol,calcium propionate, sodium nitrate, sodium nitrite, Na²EDTA, andsulfites including but not limited to sulfur dioxide, sodium bisulfite,and potassium hydrogen sulfite.

Release-controlling polymers may be included in the various embodimentsof the solid dosage compositions incorporating compounds according tothis disclosure. In one embodiment, the release-controlling polymers maybe used as a tablet coating. In other embodiments, including but notlimited to bilayer tablets, a release-controlling polymer may be mixedwith the granules and other excipients prior to the formation of atablet by a known process including but not limited to compression in atablet mold. Suitable release-controlling polymers include but are notlimited to hydrophilic polymers and hydrophobic polymers.

Suitable hydrophilic release-controlling polymers include, but are notlimited to, cellulose acetate, cellulose diacetate, cellulosetriacetate, cellulose ethers, hydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,nitrocellulose, crosslinked starch, agar, casein, chitin, collagen,gelatin, maltose, mannitol, maltodextrin, pectin, pullulan, sorbitol,xylitol, polysaccharides, ammonia alginate, sodium alginate, calciumalginate, potassium alginate, propylene glycol alginate, alginate sodiumcarmellose, calcium carmellose, carrageenan, fucoidan, furcellaran,arabicgum, carrageensgum, ghaftigum, guargum, karayagum, locust beangum,okragum, tragacanthgum, scleroglucangum, xanthangum, hypnea, laminaran,acrylic polymers, acrylate polymers, carboxyvinyl polymers, copolymersof maleic anhydride and styrene, copolymers of maleic anhydride andethylene, copolymers of maleic anhydride propylene or copolymers ofmaleic anhydride isobutylene), crosslinked polyvinyl alcohol and polyN-vinyl-2-pyrrolidone, diesters of polyglucan, polyacrylamides,polyacrylic acid, polyamides, polyethylene glycols, polyethylene oxides,poly(hydroxyalkyl methacrylate), polyvinyl acetate, polyvinyl alcohol,polyvinyl chloride, polystyrenes, polyvinylpyrrolidone, anionic andcationic hydrogels, and combinations thereof.

The invention can also include compositions that can be created as apowder that can be added to food items, as a baked good (e.g., ascookies and brownies), and as a concentrate. The concentrate can beadded to water or another ingestible liquid to create a nutritionalbeverage. The nutritional supplement is typically contained within aone-serving or multiple serving container such as a package, box,carton, wrapper, bottle or can. Where the nutritional supplement isprepared in the form of a concentrate that can be added to and mixedwith a beverage, a bottle or can be used for packaging the concentrate.The nutritional supplement can also include water.

III. Methods of Increasing Muscle Protein Synthesis

The present invention also provides methods for increasing muscleprotein synthesis, increasing muscle strength, increasing musclefunction, or a combination thereof. The method comprises administeringto a subject a combination comprising a plurality of essential aminoacids and optionally non-essential amino acids and/or nutrients.Suitable combinations and formulations for administration are describedabove in Section I and Section II, respectively. Suitable subjects mayinclude a human, a livestock animal, a companion animal, a laboratoryanimal, or a zoological animal. In a preferred embodiment, a subject ishuman. The magnitude of response may depend, in part, on the dose given,the exact combination of amino acids, the physiological condition and/orage of the subject, and/or the timing of administration in relation toperformance of exercise.

Muscle mass and function are progressively lost with aging, so that byage of 60 many otherwise healthy human subjects have reached a thresholdwhere function begins to be affected. In an aspect, administering acombination of the invention to any healthy subject may increase muscleprotein synthesis, increase muscle strength, increase muscle function,or a combination thereof, though the magnitude of the response may varydepending on age and/or the physiological condition of the subject.

The loss of body protein frequently accompanies illness, whether theillness is short term or long term. In another aspect, administering acombination of the invention to a subject that lost body protein as aresult of illness may increase muscle protein synthesis, increase musclestrength, increase muscle function, or a combination thereof in thesubject. In another aspect, administering a combination of the inventionto a subject at risk of or experiencing muscle atrophy may increasemuscle protein synthesis, increase muscle strength, increase musclefunction, or a combination thereof. Non-limiting examples of subjects atrisk of or experiencing muscle atrophy include humans with a sedentarylifestyle, people with seated jobs, medical conditions that limitmovement, subjects that are bedridden, subjects who are away fromEarth's gravity, subjects with an injury to a nerve that connects tomuscle, and subjects at risk f or diagnosed with a disease affecting thenerves that control muscles. Non-limiting examples of diseases,disorders or conditions in which muscle atrophy occurs includes polio,Guillan-Barre syndrome, chronic obstructive pulmonary disease,congestive heart failure, acute coronary syndrome, chronic heartfailure, cardiac cachexia, cancer, sarcopenia, spinal cord injury,osteoarthritis, arthritis, stroke, malnutrition, muscular dystrophy(e.g. Becker, congenital, Duchenne, Distal, Emery-Dreifuss,Facioscapulohumeral, Limb-Girdle, Oculopharyngeal), spinal muscularatrophy (e.g. ALS, motor neuron disease, infantile progressive spinalmuscular atrophy, intermediate spinal muscular atrophy, juvenile spinalmuscular atrophy, adult spinal muscular atrophy), inflammatorymyopathies (e.g. dermatomyositis, polymyositis), diseases of theperipheral nerve (e.g. Carcot-Marie tooth disease, Dejerine-Sottasdisease, Friedreich's ataxia), diseases of the neuromuscular junction(Myasthenia gravis, Lambert-Eaton syndrome), metabolic diseases of themuscle (acid maltase deficiency, carnitine deficiency, carnitinepalmityl transferase deficiency, Debrancer enzyme deficiency, Lactatedehydrogenase deficiency, Mitochondrial deaminase deficiency,Phophorylase deficiency, Phosphofructokinase deficiency,Phosphoglycerate kinase deficiency), other myopathies (central coredisease, hyperthyroid myopathy, myotonia congenita, myotubular myopathy,paramytonia congenita, periodic paralysis-hypokalemic-hyperkalemic), orany other disease or condition that requires a subject to be immobilizedor on bed rest, especially chronic heart failure and cardiac cachexia.

Suitable subjects may be of any age, including at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 8, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, at least 100 or more years of age. A subject may also beless than one year of age.

In some embodiments, a method of the invention comprises administeringto a subject a combination comprising phenylalanine, valine, threonine,tryptophan, methionine, leucine, isoleucine, lysine, and histidine in aw/w ratio of about 4:about 6:about 5:about 2:about 2:about 25:about7:about 10:about 1, respectively. In other embodiments, a method of theinvention comprises administering to a subject a combination comprisingphenylalanine, valine, threonine, tryptophan, methionine, leucine,isoleucine, lysine, and histidine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1,respectively wherein the amino acids are at least about 80% of the totalweight of the combination. In other embodiments, a method of theinvention comprises administering to a subject a combination comprisingphenylalanine, valine, threonine, tryptophan, methionine, leucine,isoleucine, lysine, histidine and citrulline in a w/w ratio of about4:about 6:about 5:about 2:about 2:about 25:about 7:about 10:about1:about 8, respectively. In other embodiments, a method of the inventioncomprises administering a combination comprising phenylalanine, valine,threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine and citrulline in a w/w ratio of about 4:about 6:about 5:about2:about 2:about 25:about 7:about 10:about 1:about 8, respectively,wherein the ten amino acids are at least about 95% of the total weightof the combination. In other embodiments, a method of the inventioncomprises administering a combination comprising phenylalanine, valine,threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively. In an exemplary embodiment a method of theinvention comprises administering a combination listed in Table A, TableB, or Table C. In preferred embodiments, a combination of the inventionis administered to a subject with chronic heart failure or cardiaccachexia.

In an exemplary embodiment, a method of the invention comprisesadministering to a subject a combination comprising phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively, wherein the subject has chronic heart failure.

In an exemplary embodiment, a method of the invention comprisesadministering to a subject a combination comprising phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively, wherein the combination further comprisesbiotin, omega-3 fatty acids or a combination thereof, and wherein thesubject has chronic heart failure.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination comprising phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively, wherein the combination further comprisesbiotin, omega-3 fatty acids or a combination thereof, and wherein thesubject has chronic heart failure and is at least 50 years old, morepreferably at least 60 years old, even more preferably at least 65 yearsold.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table A, wherein thesubject has chronic heart failure.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table A, wherein thesubject has chronic heart failure and is at least 50 years old, morepreferably at least 60 years old, even more preferably at least 65 yearsold.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table B, wherein thesubject has chronic heart failure.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table B, wherein thesubject has chronic heart failure and is at least 50 years old, morepreferably at least 60 years old, even more preferably at least 65 yearsold.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination comprising phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively, wherein the subject has cardiac cachexia.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination comprising phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively, wherein the combination further comprisesbiotin, omega-3 fatty acids or a combination thereof, and wherein thesubject has cardiac cachexia.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination comprising phenylalanine,valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine,histidine, citrulline and L-carnitine in a w/w ratio of about 4:about6:about 5:about 2:about 2:about 25:about 7:about 10:about 1:about8:about 3, respectively, wherein the combination further comprisesbiotin, omega-3 fatty acids or a combination thereof, and wherein thesubject has cardiac cachexia and is at least 50 years old, morepreferably at least 60 years old, even more preferably at least 65 yearsold.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table A, wherein thesubject has cardiac cachexia.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table A, wherein thesubject has cardiac cachexia and is at least 50 years old, morepreferably at least 60 years old, even more preferably at least 65 yearsold.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table B, wherein thesubject has cardiac cachexia.

In another exemplary embodiment, a method of the invention comprisesadministering to a subject a combination listed in Table B, wherein thesubject has cardiac cachexia and is at least 50 years old, morepreferably at least 60 years old, even more preferably at least 65 yearsold.

The frequency of administration may be once, twice, three times or moredaily, or once, twice, three times or more per week or per month, asneeded as to produce the desired effect. The amount administered to thesubject can and will vary depending upon the subject (e.g. age, weight,health status), and the particular mode of administration. Those skilledin the art will appreciate that dosages may also be determined withguidance from Goodman & Goldman's The Pharmacological Basis ofTherapeutics, Tenth Edition (2001), Appendix II, pp. 475-493, and thePhysicians' Desk Reference.

In some embodiments, a combination of the invention may be at least 5grams in weight. For example, a combination may be at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20 grams or more in weight. Inother embodiments, a combination of the invention may be about 5 toabout 10 grams, about 10 to about 15 grams, about 15 to about 20 grams,about 5 to about 15 grams, about 10 to about 20 grams, or about 5 toabout 20 grams in weight. In a preferred embodiment, a combination ofthe invention may be about 7 to about 15 grams in weight.

An increase in muscle protein synthesis may be measured by any methodknown in the art. For example, rates of protein synthesis and breakdownin muscle and blood have been measured for many years usingradioactively labeled amino acids or amino acids labeled with stableisotopes. See for example, Yale J Biol Med 1997; 70(1): 65-76,incorporated herein by reference. Measurement of 3-methyhistidineexcretion in urine or arteriovenous difference in 3-methylhistidine isused as a measure of the unidirectional rate of muscle proteinbreakdown. Total body skeletal mass can be measured using dual-energyX-ray (DEXA) absorptiometry or by CT; see, for example, Am J Clin Nutr2002; 76:378-83, and J Appl Physiol 1985 incorporated herein byreference. Methods for measuring muscle strength or function are alsowell known in the art. For example, change in muscle strength andfunction can be assessed using basic standardized tests as described inB J Nutr 2012; 108: S88-93 or in Lu et al. (2012) “Strength andFunctional Measurement for Patients with Muscular Dystrophy” MuscularDystrophy, Dr. Madhuri Hegde (Ed.), each hereby incorporated byreference.

In some embodiments, a method of the invention may increase muscleprotein synthesis by at least 2-fold. For example, muscle proteinsynthesis may increase at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, at least 10-fold or more followingadministration of a combination of the invention. In some embodiments, amethod of the invention may increase muscle strength by at least 2-fold.For example, muscle strength may increase at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, at least 10-fold or morefollowing administration of a combination of the invention. In someembodiments, a method of the invention may increase muscle function byat least 2-fold. For example, muscle function synthesis may increase atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, atleast 10-fold or more following administration of a combination of theinvention. In other embodiments, a method of the invention may increasemuscle protein synthesis, muscle strength and muscle function, or acombination thereof. Methods for measuring muscle protein synthesis,muscle strength and muscle function are known in the art and furtherdetailed in the Examples.

EXAMPLES

The following examples illustrate various iterations of the invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention. Allpatents, applications, published applications and other publications areincorporated by reference in their entirety.

Example 1 Combinations of Essential Amino Acids Stimulate Muscle ProteinSynthesis without Exercise

Applicants have surprisingly found that functional improvements indisabled and/or elderly patients can be achieved with a dietarysupplement comprised of a combination of essential amino acids (EAAs),even in the absence of an exercise program. Stimulation of muscleprotein synthesis is the metabolic basis for improvements in functionaland metabolic functions of muscle. The studies herein demonstrate thatonly EAAs are needed to stimulate muscle protein synthesis, therebyreducing the amount of amino acids that must be given to elicit aneffective response.

As shown in FIG. 1, anabolic resistance to amino acids in elderlypatients can be overcome by using a supplement that contains a highproportion of leucine (e.g. about 40% of total EAAs). In theseexperiments, net protein synthesis was measured in the leg muscle ofelderly volunteers over the age of 65 years before and after ingestionof 6.7 g of a mixture of EAAs in the profile in whey protein or amixture of the same amount of EAAs with 40% leucine.

After determining the transport rates of the individual EAAs intomuscle, a combination of EAAs was formulated that delivered each aminoacid intracellularly in proportion to the requirement for that aminoacid as a component of muscle protein. The result is a formulation(Table C) that is much more effective in stimulating muscle proteinsynthesis than a high-quality protein such as whey or an intact foodsource of a high quality protein such as a beef patty (FIG. 2).

FIG. 3 shows that the improvement in muscle protein synthesis observedafter administration of the EAA formulation in Table C translates toimprovements in practical outcome measures. For example, the losses infunctional capacity that result from enforced bed rest in both young andold were almost completely ameliorated by a daily supplement of the EAAmixture. Further, EAA supplementation of free-living elderly volunteersfor 16 weeks increased strength and functional capacity (Borsheim E, Q UBui, S Tissier, H Kobayashi, A A Ferrando, R R Wolfe “Effect of aminoacid supplementation on muscle mass, strength and physical function inelderly” (2008) Clin Nutr 27(2):189-195).

A high-leucine/protein mixture was also able to overcome anabolicresistance to conventional supplement intake in the highly-inflammatorystates of cancer, and in patients recovering from hip-replacementsurgery. Furthermore, the effectiveness of the EAA formulation is notreduced by insulin resistance.

One important aspect of the present invention is that it shows asignificant interaction with exercise. Even in young, healthyindividuals resistance exercise induces only a modest stimulation ofmuscle protein synthesis (FIG. 4). The magnitude of response to exerciseis less than the response to amino acid intake alone (FIG. 4). However,prior exercise “primes” the muscle to the anabolic action of aminoacids, such that there is an additive effect of exercise plus aminoacids. Thus, whereas exercise protocols alone may have limited successin patients with heart failure because of anabolic resistance of muscle,the combination of some level of activity with nutrient therapy willresult in a synergistic interaction that produces a significantlygreater response than from the exercise alone or the essential aminoacid mixture alone.

Example 2 Citrulline Supplementation Stimulates Nitric Oxide Productionand Increased Plasma Arginine Levels

Preferred formulations of the invention incorporate an optimizedproportion of citrulline, the precursor of the amino acid arginine.Arginine is derived from both endogenous and dietary sources. Undernormal conditions, the body produces sufficient arginine to satisfymetabolic requirements and hence is not be necessary in a mixture ofEAAs to stimulate muscle protein synthesis. However, in certain clinicalcircumstances including in elderly with heart failure, endogenousproduction is inadequate to meet all demands. We have recently foundthat arginine synthesis is decreased in elderly with heart failurebecause of a reduced availability of citrulline.

A deficiency in arginine may result in a variety of metabolicconsequences, including a reduced rate of muscle protein synthesis,impaired immune function, and decreased synthesis of nitric oxide (NO).In clinical circumstances in which de novo synthesis of arginine fromcitrulline may be inadequate, dietary supplementation can be aneffective approach. Published studies have shown dietary supplementationwith arginine reduces blood pressure and improves right ventricularfunction in patients with heart failure. These improvements translatedto increased duration until exhaustion on a treadmill walk. However,there are limitations in the use of supplemental arginine. Because ofhigh utilization of orally-ingested arginine by the gut and highfirst-pass clearance by the liver, dietary supplementation with arginineis only marginally effective in raising peripheral arginineavailability. Consequently, a large dose of arginine is necessary, andthis can cause significant gastric distress. Dietary supplementationwith citrulline is more effective in increasing arginine availability.In contrast to arginine, splanchnic clearance of citrulline is low, andthe renal conversion of citrulline to arginine is normally efficient. Incontrast to arginine, citrulline is well-tolerated.

FIG. 5 shows the outcome of an isotopic tracer study in which multiplestable isotope tracers enabled quantification of the rates of arginineand citrulline synthesis as well as the rate of NO production. Briefly,elderly (n=8, 77±9y, 93±4 kg) and young individuals (n=8, 27±9y, 70±5kg) ingested 3 grams of citrulline over 3 hours and plasma arginineresponse and nitric oxide (NO) synthesis rates were quantified. Notably,(1) NO synthesis was impaired in the elderly with heart failure ascompared to young, healthy subjects, (2) ingestion of 3 g of citrullineeffectively increased the concentration of arginine in the blood, and(3) citrulline ingestion stimulated production of NO. The extent ofincrease in NO production was less in elderly with heart failure than inyoung, but the increase in NO synthesis in elderly was physiologicallysignificant. Thus, oral ingestion of citrulline is very effective inincreasing peripheral arginine concentration and NO synthesis in elderlysubjects with heart failure.

Arginine plays a role in the regulation of muscle protein synthesisbeyond that of its contribution as a precursor of newly-synthesizedprotein. FIG. 6 shows the result of increasing the proportion ofarginine in the mixture of an infusion of intravenous amino acids onmuscle protein synthesis. The same amount of nitrogen was infused inboth groups fundamentally in the form of a commercially-availableintravenous formulation (Travasol), but in the arg-Travasol group extraarginine was added (and the rest of the amino acids reducedcorrespondingly).

The arginine effect on muscle protein synthesis is apparentlyindependent of its effect on NO synthesis, as arginine administrationwas effective even when given when NO synthesis was pharmacologicallyblocked. See, for example, Zhang X-J, Chinkes D L, Wolfe R R “Theanabolic effect of arginine on proteins in skin wound and muscle isindependent of nitric oxide production” (2008) Clin Nutr 27:649-656.

Taken together, these data support adding citrulline to a formulation ofEAAs in order to improve outcomes by increasing NO synthesis, as well asmuscle protein synthetic response through increased arginineavailability.

Example 3 Supplementation Improves Insulin Sensitivity

Physical and cardiac function requires adequate ATP production to enablemuscular contraction to occur. This not only involves functionalvascular reactivity to enable increased oxygen and substrate delivery tocontracting muscles, but also appropriate regulation of substratemetabolism and unimpaired oxidative capacity of mitochondria. Insulinresistance occurs commonly in elderly individuals with heart failure,particularly those who are obese, and the extent of insulin resistanceis related to the deposition of fat in the liver. The EAA combination ofTable C reduced hepatic fat (FIG. 7), which was related to animprovement in insulin sensitivity.

Example 4 Supplementation Increases the Number and Functional Capacityof Mitochondria in Subjects

Appropriate regulation of energy substrate metabolism not only involvesrelatively normal insulin sensitivity, but also adequate number andfunctional capacity of mitochondria. One aspect of mitochondrialfunction is the rate of production of mitochondrial proteins. An EAAcombination comprising phenylalanine, valine, threonine, tryptophan,methionine, leucine, isoleucine, lysine, and histidine stimulatesmitochondrial proteins in skeletal muscle in human subjects (Bohe J, ALow, R R Wolfe, M J Rennie “Human muscle protein synthesis is modulatedby extracellular but not intracellular amino acid availability: A doseresponse study” (2003) J Physiol 552:315-324); and leucine has recentlybeen shown to stimulate mitochondrial biogenesis. In addition toadequate protein content in mitochondria, it is necessary thatsubstrates, particularly long-chain fatty acids, are not limited intheir ability to enter the mitochondria, or in their complete oxidationonce inside the mitochondria. The capacity to oxidize fatty acids isreduced in patients with major inflammatory responses due to reducedcarnitine palmitoyltransferase 1 (CPT1) activity. Unimpaired capacity tooxidize fatty acids is important because the energy to performlow-intensity exercise, including activities of daily living thatindividuals with heart failure often struggle with, is provided by theoxidation of fatty acids. As shown in FIG. 8, CPT-1 activity declineswith advancing age and with oxidative stress in rodent hearts, andtreatment of older hearts with propionyl-L-carnitine can restore CPT-1activity to that of young hearts. Taken together, these data supportadding L-carnitine to a formulation of EAAs in order to improve outcomesby increasing fatty acid oxidation.

What is claimed is:
 1. A method for increasing muscle protein synthesis,increasing muscle strength, increasing muscle function, or a combinationthereof, the method comprising administering to a subject with chronicheart failure, cardiac cachexia or a combination thereof a combinationcomprising phenylalanine, valine, threonine, tryptophan, methionine,leucine, isoleucine, lysine, histidine, citrulline and L-carnitine in aw/w ratio of about 4:about 6:about 5:about 2:about 2:about 25:about7:about 10:about 1:about 8:about 3, respectively, and biotin, omega-3fatty acids, or a combination thereof.
 2. The method of claim 1, whereinthe combination is the combination listed in Table B.
 3. The method ofclaim 1, wherein the subject is a human.
 4. The method of claim 1,wherein the subject is at risk of muscle atrophy.
 5. The method of claim1, wherein the subject is also diagnosed with a disease or conditionselected from the group consisting of polio, Guillan-Barre syndrome,congestive heart failure, acute coronary syndrome, cancer, sarcopenia,spinal cord injury, osteoarthritis, arthritis, stroke, malnutrition,muscular dystrophy, spinal muscular atrophy, inflammatory myopathy, adisease of the peripheral nerve, a disease of the neuromuscularjunction, and a metabolic disease of the muscle.